Sodium salt of 3-(4-cinnamyl-1-piperazinyl)-imino-methyl rifamycin SV

ABSTRACT

Sodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV was synthesized. The compound shows high activity against Gram-positive and Gram-negative microorganims, Mycobacterium tuberculosis (including atypical and rifampicin resistant) and may be used in the medical practice. The sodium salt has formula (II). The process for preparation of the sodium salt consists of reacting equimolar quantities of 3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV and sodium ascorbate with addition of 30% methanol solution of sodium methylate, followed by filtration and removement of the solvent by distillation under reduced pressure. The compound can also be obtained from the sodium salt of 3-formil rifamycin SV, which is reacting with N 1 -amino-N 4 -cinnamypiperazin in medium of inert solvent at room temperature.

Sodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV wassynthesized.

The compound shows high activity against Gram-positive and Gram-negativemicroorganisms, Mycobacterium tuberculosis (including atypical andrifampicin resistant) and may be used in the medical practice. Thesodium salt has the following formula:

The process for preparation of the sodium salt consists of reactingequimolar quantities of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV and sodium ascorbate with addition of 30% methanol solutionof sodium methylate, followed by filtration and removement of thesolvent by distillation under reduced pressure.

The compound can also be obtained from the sodium salt of 3-formilrifamycin SV, which is reacting with N¹-amino-N⁴-cinnamypiperazine inmedium of inert solvent at room temperature.

TECHNICAL FIELD

The invention relates to the sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl-rifamycin SV and process ofpreparation. The sodium salt shows high activity against Gram-positiveand Gram-negative microorganisms, Mycobacterium tuberculosis, (includingatypical and Rifampicin resistant) and therefore may be used in themedical practice.

BACKGROUND OF THE INVENTION

The rifamycins are group of antibiotics with high antibacterial activityand they have a wide spectrum of application in the treatment ofMycobacterium infections. Rifampicin is the best known representative ofthe group of rifamycins.

In document BG No. 36006 (U.S. Pat. No. 4,193,920) are described newazomethyn derivatives of Rifamycin SV with general formula I,

These compounds display high activity against Gram-positive andGram-negative microorganisms and Mycobacterium tbc. This activity isanalogical and in some cases is higher than that of Rifamycin. Thedocument BG No. 87451 (U.S. Pat. No. 5,095,108 (1992)) describes aprocess for preparing the insoluble crystal form of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV (compound,designed as T-9 in the first quoted BG document). The in vivoinvestigations of the compound show a higher therapeutic effect incomparison with this of Rifampicin. The therapeutic activity of T-9 indoses 10 mg/kg in generalized tuberculosis in test animals shows fullorgan sterilization after 60 days of treatment, while a similartherapeutic effect is achieved with Rifamificyn in doses of 80 mg/kg.The compound presents considerably longer serum half life thanRifampicin (T_(½): 31˜34 hours) tested on animals.

The acute toxicity of T-9 in mice is 4000 mg/kg, while this ofRifampicin is 1500 mg/kg. This indicates the compound T-9 as the bestperspective among the derivatives of Rifamicyn SV, described in BG No.36006.

DISCLOSURE

The present invention is concerned with the sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV which is new, anda process for its preparation. The new sodium salt provided by thepresent invention is compound of the general formula II:

The new sodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV has increased antibacterial activity and low toxicity incomparison with the compound “T-9”. The new sodium salt has thefollowing advantages: good water solubility and higher bioavalabilityand possibility for administration as injectable formulation.

The synthesis of the water soluble sodium salt of “T-9” provides betterpossibilities for administration of the new salt as well in parenteralas in peroral pharmaceutical formulations.

The in vitro studies of the activity of sodium salt (designated T-1 1)in comparison with the initial compound T-9 show good antibacterialactivity against the Gram-positive microorganisms including aerobic,anaerobic and Mycobacterium (typical and atypical). The pharmacokineticproperties of the known compound T-9 are improved in the salt—the sodiumsalt has better resorbtion characteristics resulting from its betterwater solubility and it maintains higher serum levels. The sodium salthas clearly expressed depo activity and secures the maintenance ofconstant theurapeutical concentrations in the organism for a longerperiod of time: 40˜50 hours in mice and rats, and about 100 hours inrabbits (Table 1).

TABLE 1 Non-model pharmacokinetic parameters after use of T-11 and T-9(base 20 mg/kg) in mice, rats and rabbits Method C_(max) CL ofApplication mg/ T_(max) T_(1/2) AUC ml · min/ V Species of Animals ml hh mg · h/ml kg J/kg Mice T-11 i.v 50 1 39.08 3616.7 0.092 0.312 p.o 14.48 38.02 977.45 0.092 0.303 T-9 p.o 12.4 8 33.70 734.69 0.092 0.270 RatsT-11 p.o 9.20 2 20.04 671.352 T-9 p.o 8.20 6 21.18 543.144 Rabbits T-11p.o 2.71 2 28.04 298.8 T-9 p.o 2.75 2 56.63 232.6

The advantages of the newly-synthesed sodium salt are the good watersolubility and the possibility of its application in the form ofinjection solutions (which is impossible with the unsoluble startingcompound T-9), good stability of the obtained water solutions, fasterresorbtion and better pharmacokinetic properties in animal tests.

The sodium salt of 3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SVcan be easily obtained by the following two methods:

Method A.

In this method the sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV is prepared byreacting equimolar amount of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV and of appropriate organic or inorganic base, containingsodium, in aqueous medium or in organic solvent medium. Preferably thereaction can be carried out in the presence of small amount of sodiumascorbate as an antioxidizing agent to prevent the transformation of thephenol structures in the rifamycin derivative into quinine-structures.When the reaction is carried out in an aqueous medium, diluted solutionof sodium salt is used as a base and when using sodium alcoholates(sodium methylate, sodium ethylate, sodium isopropilate), the reactionmixture is prepared by adding the corresponding alcohol (methanol,ethanol and the like). More preferably is the use of sodium methylatediluted with methanol or ethanol. The solvent was removed in vacuum (ifit is alcoholate) and the resulting solid sodium salt is distillated oris lyophilized (if the reaction is carried out in aqueous medium).

The sodium salt may be isolated using distillation of the solving agentin vacuum (when effecting the reaction in an alcoholic medium), or usinglyophilization (when effecting the reaction in water).

The yield of the sodium salt is practically quantitative and with highpurity. The obtained the sodium salt may be purified byrecrystallization in a suitable solvent.

Method B.

Proceeding as described in Method A is prepared a sodium salt of3-formil rifamycin SV which subsequently is reacting withN¹-amino-N⁴-cinnamylpiperazine in medium of an inert solvent at roomtemperature. The solvent was evaporated by distillation in vacuum andthe residual product was purified by recrystallization in suitablesolvent.

The following examples are given to illustrate more clearly the presentinvention.

EXAMPLE 1

4,62 g (0,005 g/moll) of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV is suspended in 40 ml methanol and 0,1 g sodium ascorbatedissolved in 1 ml water is added. To the obtained suspension 0,9 ml of30% methanol solution of sodium methylate (equimolar quantity) is added.The suspension immediately turns into a solution because of theformation of the sodium salt. The reaction mixture is filtered to removethe unsolved sediment of sodium ascorbate, and the solvent isconcentratrated in vacuum and dried to give the soduim salt as adark-red solid powder. This product has high purity and the yield ispractically quantitative. The crude product opptionally may berecrystallized by boiling in isopropanol to give the sodium salt as redcrystal solid (80% yield), soluble in water, methanol, ethanol, acetoneand unsoluble in isopropanol.

The absorption in UV-spectrum and visible area of 0,001% methanolsolution of the product in the interval 200-800 nm in cuvette, withlayer with 1 cm, has a maximums wavelength of 251, 338 and 480 nm.

Analysis: C₅₁H₆₃N₄ NaO₁₂ M.m 947,03

% content of Na (theoretical): 2,43

% content of Na (determined by the method of atomic-absorptionanalysis): 2,35

EXAMPLE 2

In a manner analogous to that described in Example 1 and substitutingthe methanol with ethanol the sodium salt was obtained.

EXAMPLE 3

In a manner analogous to that described in Example 1 and substitutingthe methanol with isopropanol the sodium salt was obtained.

EXAMPLE 4

In a manner analogous to that described in Example 1 and subsituting the30% methanol solution of sodium methylate with equimolar quantity of drysodium methylate the sodium salt was obtained.

EXAMPLE 5

4,62 g (0,005 g/moll) of 3-(4-cinnamyl-1-piperazinyl)-iminomethylrifamycin SV is suspended in 100 ml of water, and 5 ml 1N solution ofsodium base is added to the suspension for two to three hours withvigirous stirring. A gradual transformation of the suspension into asolution is observed, due to the formation of the soluble sodium salt.The reaction mixture is filtered and lyophilized to give the sodium saltas dark red powder (quantitative yield).

Analysis: C₃₈H₄₆NNaO₁₃

% content of Na (theoretical): 3,07

% content of Na (determined by the method of atomicabsorbationanalysis): 3,05

EXAMPLE 6

5,23 g (0,007 g/moll) of the obtained product is dissolved in 50 mltetrahydrofuran and to the solution with continuous stirring 1,52 g(0,007 g/moll) N¹-amino-N⁴-cinnamylpiperazine, dissolved in 20 mltetrahydrofuran, is added. The reaction mixture is stirred at roomtemperature for 2 h. The solvent is then redistillated under reducedpressure. The obtained sodium salt of3-(4-cinnamyl-1-piperazinyl)-iminomethyl rifamycin SV is recrystallizedin isopropyl alcohol. Yield of 4,97 g (75%)

The product is identical to this obtained by the procedure in Example 1.

What is claimed is:
 1. Sodium salt of3-(4-cinnamyl-1-piperazinyl)-imino-methyl rifamycin SV of the formula:


2. A method for the treatment of diseases caused by bacterial infectionscomprising administering an effective amount of a sodium salt of3-(4-cinnamyl-1-piperazinyl)-imino-methyl rifamycin SV.
 3. Apharmaceutical formulation for the treatment of diseases caused bybacterial infections comprising a unit dose of a sodium salt of3-(4-cinnamyl-1-piperazinyl)-imino-methyl rifamycin SV and apharmaceutical excipient.